Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/31895
標題: 疫病菌及&;#63800;疫病菌有性世代之生物及生&;#63972;特性
Biological and Physiological Characterization of Sexual Reproduction in Phytophthora and Peronophythora
作者: 林玫珠
Lin, Mei-Ju
關鍵字: Phytophthora capsici
青椒疫病
Peronophythora litchii
mitochondrion-zoospor fusion
mitochondria genome
Phytophthora colocasiae
Homothallic
sexual reproduction
mating type
荔枝露疫病
粒線體-游走孢子融合
粒線體基因組
芋頭疫病菌
同宗交配型菌株
有性世代
交配型
出版社: 植物病理學系所
摘要: 在疫病菌 (Phytophthora) 屬中,大多&;#63849;的種 (species) 為病原菌,他們引起主要的&;#63867;食作物、蔬菜及水果發生病害,成為生產過程的重要限制因子。有些種為同宗交配型 (homothallic),單獨培養時可產生&;#63772;孢子,有些種為&;#63842;宗交配型(heterothallic),必須在同種或&;#63847;同種間的A1及A2交配型進&;#64008;配對才會產生&;#63772;孢子;然而,有性繁殖過程中所發生的遺傳重組,會使疫病菌創造出新的遺傳變&;#63842;及新的病原性的個體。芋頭疫病菌 (Phytophthora colocasiae) 引起芋&;#63854;疫病及塊莖腐&;#63774;,2007&;#63886;之前,鮮少有研究證明關於芋頭疫病菌在土壤中存活的情形。因此,本研究對台灣芋頭疫病菌重新調查其交配型 (mating type),調查過程中,由台灣中部&;#63974;病芋&;#63854;上分&;#63978;出7個同宗交配型的芋頭疫病菌株;當這些同宗交配型的菌株接種在活的芋頭&;#63854;柄組織,放入土壤中經過一個月,芋頭&;#63854;柄上產生大&;#63870;的&;#63772;孢子,表示在自然界中,這些&;#63772;孢子可能做為芋頭疫病菌的存活構造,同時成為遺傳變&;#63842;的&;#63789;源。 已知疫病菌屬的有性繁殖是藉由賀爾蒙 (hormone) &;#63789;進&;#64008;調控,然而交配型的遺傳特性至今仍&;#63847;清楚。本研究以青椒疫病菌 (Phytophthora capsici) 為材&;#63934;,發展出有效&;#63841;的&;#63993;線體 (mitochondrion) 或細胞核 (nucleus) 與游走孢子融合的系統,用以研究交配型的遺傳控制。將A1交配型的&;#63993;線體轉移到A2交配型的游走孢子中,獲得的&;#63993;線體雜交株 (mitochondrial hybrids) 可在單獨培養時產生&;#63772;孢子,表示交配型由&;#63993;線體上的基因所控制;經RFLP分析其&;#63993;線體DNA,結果顯示&;#63993;線體雜交株中確實帶有轉移的外&;#63789;&;#63993;線體DNA,證明A1交配型菌株的&;#63993;線體的確成功轉移進入A2交配型菌株的游走孢子內。藉著&;#63993;線體與游走孢子融合的方法,成功將&;#63993;線體轉移的比&;#63841;在0.8 到1.0% 之間,相較於&;#63993;線體與原生質體融合 (mitochondrion-protoplast fusion) 的方法,&;#63993;線體與游走孢子融合的方法&;#63745;為簡單而且成本較低。 我們的結果顯示荔枝&;#63800;疫病菌 (Peronophythora litchii) 與疫病菌相同具有產生α賀爾蒙的能&;#63882;,產生的賀爾蒙可&;#63999;激青椒疫病菌的A1與A2交配型菌株產生&;#63772;孢子。將游走孢子融合的方法應用於&;#63847;同屬之間,把荔枝&;#63800;疫病菌的&;#63993;線體或細胞核與青椒疫病菌的游走孢子進&;#64008;融合,獲得的&;#63993;線體雜交株具有與荔枝&;#63800;疫病菌相同的同宗交配型,所產生的&;#63772;孢子型態大部分與提供&;#63993;線體的荔枝&;#63800;疫病菌相似;然而,細胞核雜交株 (nuclear hybrids) 的交配型&;#64038;與青椒疫病菌相同。這些結果表示在疫病菌及&;#63800;疫病菌的交配型的基因&;#64038;編碼在&;#63993;線體DNA上而非細胞核DNA上。由Heteroduplex mobility assay (HMA) 分析顯示&;#63993;線體雜交株中帶有外&;#63789;移轉的&;#63993;線體DNA,再一次證實疫病菌之交配型由&;#63993;線體上的基因所控制,而&;#63800;疫病菌調控有性繁殖的過程也與疫病菌相似。 二個相同親本&;#63789;源但&;#63847;同交配型的二菌株Pca1-Cn5 A1及PcaA1-Cn24 A2,將其&;#63993;線體基因組進&;#64008;全解序,得到的基因組大小分別為38417 bp及38414 bp,它們的A+T含&;#63870;為78.2%。&;#63993;線體基因組中包含61個已知功能的基因及&;#63953;個未知功能的基因,這些已知的功能的基因,包含有18個為電子傳遞相關蛋白、16個為核醣蛋白、2個為核醣RNA與25個轉移RNA。在A1與A2交配型的二菌株&;#63993;線體基因組間有9個單核&;#33527;酸多型性 (single nucleotide polymorphisms, SNPs),由這些結果推&;#63809;A1與A2交配型基因位於&;#63847;同的&;#63993;線體DNA上。綜合本研究之結果顯示疫病菌的交配型基因位於&;#63993;線體DNA上,顯然這九個SNPs可能&;#63851;與並決定交配型,然而這些SNPs中,與調控交配型相關的機制仍需&;#63745;進一步的研究才能釐清。
Most species of Phytophthora are pathogens and diseases caused by them are an important limiting factor in the production of staple food crops, vegetables and fruits. Some species of Phytophthora are homothallic which produce oospores in single culture, and the others are heterothallic which produce normal oospores when A1 and A2 mating types of the same or different species are paired in culture. Sexual recombination in Phytophthora will create new genetic variation and produce unusually pathogenic individuals. Before 2007, survival of Phytophthora colocasiae, the causal organism of taro leaf blight and corm rot, in soil is poorly documented. During our resurvey of the mating type distribution of P. colocasiae in Taiwan, 7 new homothallic isolates were obtained from diseased taro leaves in central Taiwan. The homothallic isolates produced abundant oospores in live tissue of taro petioles on soil, indicating the possibility of oospores as a survival structure and the source of genetic variation in certain areas in nature. In Phytophthora, hormone regulation is known to be involved in sexual reproduction. However, the genetic characteristics of mating types are not yet clear so far. We developed effective mitochondrion- and nucleus-zoospore fusion systems for the study of genetic control of mating type in Phytophthora capsici. Transfer of A1 mitochondria to A2 zoospores resulted in mitochondrial hybrids capable of producing oospores in single cultures. Asexual progeny from the mitochondria hybrids consisted of both A1 and A2 mating types, indicating mitochondrial gene control of mating type. The genetic analysis using RFLP of mitochondrial DNA showed that the polymorphic fragment of mitochondrial donor was present in mitochondria hybrid confirming the successful transfer of A1 mitochondria to A2 zoospores. The ratio of successful mitochondrial transfer ranged from 0.8 to 1.0% by mitochondrion-zoospore. It is simple and inexpensive in comparison with mitochondrion-protoplast fusion. The method was also used in the intergenus fusions between Peronophythora litchii organelles and Phytophthora capsici zoospores. Our results showed that Pe. litchii has the ability to produce α hormones and stimulate both A1 and A2 types of P. capsici to form oospores. The mitochondrion-zoospore fusion tests showed that the mitochondrial hybrids behaved as homothallic just like Pe. litchii, and the morphology of oospores produced from them was mostly similar to mitochondrial donor Pe. litchii. However, the mating type of nuclear hybrids was the same as P. capsici. These results indicated that mating type genes are encoded in mitochondrial DNA, but not in nuclear DNA in Phytophthora and Peronophythora. Moreover, the heteroduplex mobility assay (HMA) analysis also showed the presence of mtDNA of mitochondrial donor in the mitochondrial hybrid. The study confirms again the previous report of mitochondrial gene control of mating type in Phytophthora, and shows that control of sexual reproduction in Peronophythora is similar to that in Phytophthora. The entire mitochondrial genomes of PcaA1-Cn5 A1 and PcaA1-Cn24 A2 of Phytophthora capsici were sequenced. Two isolates with different mating type were originated from the same parent. The genome sizes were 38417 bp for A1 and 38414 bp for A2. Their A+T contents were 78.2%. The genomes contained 61 genes with known functions and six open reading frames were with unknown functions. The functions of these genes included 18 electron transport proteins, 16 ribosomal proteins, 2 ribosomal RNAs and 25 transfer RNAs. Nine single nucleotide polymorphisms were present when genome sequences of A1 and A2 mating type isolates were compared. The results suggest that A1 and A2 mating type genes are located in different mtDNA. Since results from this study show that mating type genes are encoded in mtDNA in Phytophthora, the nine SNPs are apparently involved in the determination of mating types. The mechanism by which these SNPs regulate the mating type remains to be investigated.
URI: http://hdl.handle.net/11455/31895
Appears in Collections:植物病理學系

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